CN103343168A - Rapid identification method for pathogenic fungi - Google Patents
Rapid identification method for pathogenic fungi Download PDFInfo
- Publication number
- CN103343168A CN103343168A CN2013103202424A CN201310320242A CN103343168A CN 103343168 A CN103343168 A CN 103343168A CN 2013103202424 A CN2013103202424 A CN 2013103202424A CN 201310320242 A CN201310320242 A CN 201310320242A CN 103343168 A CN103343168 A CN 103343168A
- Authority
- CN
- China
- Prior art keywords
- fungi
- bacterium
- peanut
- sclerotium
- described method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a rapid identification method for pathogenic fungi, which specially comprises the following steps: (1) culturing fungi on a solid culture medium; (2) taking fresh fungi, and extracting the DNA of the fungi by using a microwave method; (3) amplifying DNA fragments between ribosomal spacers of the fungi by using ITS1 and ITS4 primers through PCR (polymerase chain reaction); (4) after recovering the PCR fragments, carrying out sequence determination on the fragments; and (5) carrying out comparison on the obtained sequences by using the Blast of NCBI. According to the invention, the identification of unknown pathogenic fungi can be performed within a shorter time and under relatively simple experimental conditions, thereby facilitating the rapid diagnosis for diseases occurring in fields so as to guide the prevention and control of diseases. According to the method, through the identification on 21 kinds of pathogenic fungi on different crops, and the coincidence rate is 100% through sequence comparison. The method is not only applicable to the identification on fresh mycelia, but also is applicable to the identification on conidium and sclerotium of pathogenic fungi, and has the characteristics of rapidness and high efficiency.
Description
Technical field
The invention belongs to pathogenic fungi and identify the field, be specifically related to a kind of method of Rapid identification pathogenic fungi.
Background technology
Peanut pathogenic fungi harm peanut causes production loss, influences the quality of product.In order effectively to prevent and treat disease, need the cause of disease of Rapid identification peanut, in time specific aim is prevented and treated this disease.At present to fungi except traditional morphology is identified, in order to obtain information more accurately, generally rely on the understanding to fungal gene group DNA information, the PCR product that obtains after by the general PCR primer of fungi fungal gene group rDNA internal transcribed spacer district being increased carries out sequential analysis.The extracting method of conventional fungal DNA is: liquid culture 5-7d is obtained hypha,hyphae group filter, drying or liquid nitrogen freezing are handled the back by CTAB or the broken hypha,hyphae cell walls of SDS method, remove albumen by benzene/chloroform extracting again, obtain fungal DNA finally by crossing Virahol or ethanol sedimentation.The fungal DNA that obtains is obtained target gene fragment through pcr amplification, be connected on the T carrier by reclaiming target gene fragment after the gel electrophoresis again, and transform bacteria competent cell, the mono-clonal that the competent cell spread plate that transforms is cultivated the back acquisition is accredited as positive colony by PCR, and positive colony is submitted to through the bacterium liquid after cultivating and carried out sequential analysis.In this traditional method, the time of fungus culture is long, need the amount of hypha,hyphae more, the method spended time that fungal DNA extracts is longer, and the reagent that expends and human cost are also higher, complex steps, fungal DNA is by connection carrier behind the pcr amplification, the transform bacteria competence, and the clone of acquisition identifies also needs at least 2 days time, add the follow-up order-checking time, traditional authentication method needs the time about 2 weeks at least.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provide a kind of simple to operate, cost is low, flux is high and be applicable to the authentication method of Rapid identification pathogenic fungi.
For realizing goal of the invention, technical scheme of the present invention is:
A kind of method of Rapid identification pathogenic fungi comprises the steps:
(1) cultivates fungi to be identified, obtain radicula byssoidea, sclerotium or conidium;
(2) get above-mentioned radicula byssoidea, sclerotium or conidium and place damping fluid, carry out microwave thermal and shake after the broken wall treatment, place on ice, get supernatant liquor after centrifugal, be the fungal DNA extracting solution;
(3) utilize fungi universal primer ITS1 and ITS4 that above-mentioned fungal DNA extracting solution is carried out pcr amplification, amplified production carries out agarose gel electrophoresis, and recycling PCR reclaims test kit recovery purifying and obtains the purpose fragment;
(4) the above-mentioned purpose fragment is directly carried out sequential analysis, the nucleotides sequence that obtains is listed in the fungi sequence that NCBI website use blast search is complementary with it, and matching length is the longest, and sequence identity is that 100% fungi is judged to be the fungi of being identified under the fungi.
In the such scheme, described fungi to be identified is selected from step (1): the aspergillus flavus bacterium, the peanut sclerotium rolfsii bacterium, peanut focal spot germ, peanut reaping hook germ, the peanut alternaric bacteria, the apple sheath blight fungus, the leaf muld of tomato bacterium, gaeumannomyces graminis, lawn coin pinta bacterium, verticillium dahliae, melon didymella bryoniae, the rice green smut bacterium, dosporium cucumerinumand its, corn is bent the spore germ, the sweet potato black rot bacterium, rice blast fungus, cucumber fusarium axysporum, the hami melon wilt, the Chinese cabbage alternaria, the Chinese sorghum rhizoctonia solani, the citrus scab bacterium, Chinese ephedra beading reaping hook fungi, the pepper anthracnose bacterium, cucumber anthracnose, or cereal reaping hook fungi.
In the such scheme, the described fungi of step (1) carries out flat board or slant culture at PDA substratum or czapek's solution.
In the such scheme, the described fungi of step (1) 28 ℃ of dark cultivations on the PDA substratum were grown to 2～3cm to bacterium colony in 1～5 day, obtained described radicula byssoidea; Or described fungi 28 ℃ of dark culturing 7～15 days on the PDA substratum, obtain described sclerotium; Or described fungi 28 ℃ of dark culturing 5～7 days on czapek's solution, obtain described conidium.
In the such scheme, the described radicula byssoidea of step (2), sclerotium or conidial quality are 0.2～2mg.
In the such scheme, the described damping fluid of step (2) is the TE damping fluid, and its moiety is: 10mMTris-HCl, 1mM EDTA, pH=8.0.
In the such scheme, the shake microwave power of broken wall treatment of the described microwave thermal of step (2) is 750W, and the time of microwave treatment is 30s～5min.
In the such scheme, the described microwave thermal of step (2) is shaken after the broken wall treatment, places 5～10min on ice immediately, carries out centrifugal then.
In the such scheme, described centrifugally at room temperature carry out, centrifugal rotation speed is 10000rpm, the centrifugal time is 1～2min.
The system of above-mentioned pcr amplification is: the ITS1 primer of 10 * Taq buffer2 μ l, 10pmol and ITS4 primer each 1 μ l, fungal DNA extracting solution 2 μ l, 1U Taq DNA polymerase1 μ l, 2.5mM MgCl
21 μ l, and 2mmol/L dNTPs1 μ l complement to 20 μ l with aqua sterilisa again; Amplification condition is: 94 ℃ of 3min sex change, and 94 ℃ of 30s, 51 ℃ of 30s, 72 ℃ of 1min, 35 circulations, 72 ℃ of 7min extend.
The ultimate principle of utilization of the present invention is: utilize the shake hypha,hyphae of broken wall treatment trace of microwave thermal, fungal DNA can discharge from cell and enter damping fluid; Energy passivation DNA enzyme under EDTA among the damping fluid TE and the low temperature environment, centrifuging is removed fungal cell's relic and enzyme; The supernatant liquor that obtains utilizes to distinguish between the general PCR primer I TS1 of fungi and the fungi rrna of ITS4 and increases as the template of PCR, and the PCR product that obtains directly carries out sequencing after reclaiming; By the sequence of measuring is carried out the highest fungal DNA sequence of Blast comparison consistence in the NCBI sequence library, be the fungi that belongs to the target fungi.Among the present invention, the whole evaluation of finishing fungi only needs the time in 1 week.
Beneficial effect of the present invention: the present invention is simple to operate, cost is low, flux is high, evaluation speed is fast, reliability is high; Compare with traditional method, present method has been saved mirror time, reagent, consumptive material and manpower, is fit to peanut and other crop cause of diseases are carried out quick diagnosis, for the time has been striven in the control of fungal disease.
Description of drawings
The pcr amplification result of the DNA that Fig. 1 obtains under the time at the different microwave actions of different TE concentration for peanut focal spot mycelia, the band 1:30S10 * TE among the figure, 2:30S5 * TE, 3:30S1 * TE, 4:30s ddH
2O, 5:1min10 * TE, 6:1min5 * TE, 7:1min1 * TE, 8:1min ddH2O, 9:2min10 * TE, 10:2min5 * TE, 11:2min1 * TE, 12:2min ddH2O, 13:3min10 * TE, 14:3min5 * TE, 15:3min1 * TE, 16:3min ddH2O, 17:5min10 * TE, 18:5min5 * TE, 19:5min1 * TE, 20:5min ddH2O, the contrast of 21CK(clear water).
Fig. 2 is the pcr amplification result of the pathogenic fungi DNA of the Different Crop of employing microwave method extraction, band 1 among the figure: the apple sheath blight fungus, 2: leaf muld of tomato bacterium, 3: gaeumannomyces graminis, 4: lawn coin pinta bacterium, 5: verticillium dahliae, 6: melon didymella bryoniae, 7: paddy rice rice district germ, 8: dosporium cucumerinumand its, 9: corn is bent the spore germ, 10: sweet potato black rot bacterium, 11: rice blast fungus, 12: cucumber fusarium axysporum, 13: peanut focal spot germ, 14: hami melon wilt, 15: Chinese cabbage alternaria, 16: the Chinese sorghum rhizoctonia solani, 17: the citrus scab bacterium, 18: Chinese ephedra beading reaping hook fungi, 19: pepper anthracnose bacterium, 20: cucumber anthracnose, 21: cereal reaping hook fungi, the contrast of 22:CK(clear water).
Fig. 3 is the pcr amplification result of the different shape peanut pathogenic fungi DNA of employing microwave method extraction, band 1 among the figure, 2 is from different local peanut focal spot bacterium, 3: the aspergillus flavus spore, 4: the peanut alternaric bacteria, 5,6,7 is from different local peanut sickle-like bacteria, 8: the white thin,tough silk mycelia of peanut, 9: the white thin,tough silk sclerotium of peanut.
Embodiment
The present invention extracts the processing parameter of fungal DNA in order to obtain microwave method, is research object with peanut focal spot bacterium mycelia, has studied the parameter condition of microwave method extraction fungal DNA, and the concrete operations step is as follows:
(1) cultivate peanut focal spot bacterium: peanut focal spot bacterium is inoculated on the PDA substratum, and 28 ℃ dark cultivates 4d and grows to 2～3cm to bacterium colony, obtains hypha,hyphae.
(2) microwave method is extracted fungal DNA: utilize inoculating needle that the fresh mycelia (being hypha,hyphae) that grows on the PDA substratum is got 0.2mg in the centrifuge tube of the 1.5ml of sterilization, add 50 μ l10 * TE damping fluid, 50 μ l5 * TE damping fluid, 50 μ l1 * TE damping fluid or 50 μ l H
2O shook after several seconds, was positioned in the microwave oven 750W microwave 30s, 1min, 2min, 3min, or 5min, place 5min on ice after the taking-up immediately, get supernatant liquor after centrifugal (centrifugal rotational speed is 10000rpm, and the time is 1min) again and transfer in the fresh centrifuge tube, obtain the fungal DNA extracting solution.The component of described TE damping fluid is: 10mM ris-HCl, 1mM EDTA, pH=8.0.
(3) utilize fungi universal primer ITS1 and ITS4 that above-mentioned fungal DNA extracting solution is carried out pcr amplification, the system of pcr amplification is: the ITS1 primer of 10 * Taq buffer2 μ l, 10pmol and ITS4 primer each 1 μ l, fungal DNA extracting solution 2 μ l, 1U Taq DNA polymerase1 μ l, 2.5mM MgCl
21 μ l, 2mmol/L dNTPs1 μ l, aqua sterilisa complement to 20 μ l; Amplification condition is: 94 ℃ of 3min sex change, and 94 ℃ of 30s, 51 ℃ of 30s, 72 ℃ of 1min, 35 circulations, 72 ℃ of 7min extend.
(4) amplified production carries out agarose gel electrophoresis, the results are shown in following table 1 and Fig. 1.
Can obtain from table 1 and Fig. 1: the fresh mycelia 0.2mg of fungi of trace places TE damping fluid (1 * TE damping fluid～10 * TE damping fluid), (DNA that 30s～5min) all can obtain capacity carries out pcr amplification to carry out the short period of time microwave treatment, and amplified band is clear, illustrates that this method is applicable to the Rapid identification of pathogenic fungi.
Low-concentration buffer is little to subsequent P CR reaction influence, and the supernatant liquor behind the microwave can reduce treatment step and the loss of DNA directly as the template of PCR reaction.The present invention chooses the condition of " concentration 1 * TE damping fluid; microwave action time 30s～5min " as the condition of microwave method extraction fungal DNA among following embodiment 1 and the embodiment 2, adopts microwave method to extract fungal DNA and can save needed time of extraction fungal DNA, manpower and reagent consumption greatly.
The pcr amplification result of the DNA sample that the different TE buffer concentrations of table 1, different treatment time obtain
| Treatment time | 10XTE | 5XTE | 1XTE | H 2O |
| 30s | + | + | + | + |
| 1min | + | + | + | - |
| 2min | + | + | + | - |
| 3min | + | + | + | - |
| 5min | + | + | + | - |
Annotate: "+" expression obtains target DNA fragment; "-" expression does not amplify target DNA fragment
A kind of method of Rapid identification pathogenic fungi specifically comprises the steps:
(1) cultivate fungi to be identified: fungi to be identified is inoculated on the PDA substratum, and 28 ℃ secretly are cultured to bacterium colony and grow to 2-3cm, obtain radicula byssoidea.
(2) microwave method is extracted the DNA of fungi: utilize inoculating needle that the fresh mycelia (being above-mentioned radicula byssoidea) that grows on the PDA substratum is got 2mg in the centrifuge tube of the 1.5ml of sterilization, 1 * TE damping fluid, the 50 μ l that add, shake after several seconds, be positioned in the microwave oven, 750W microwave 2min places 7min on ice immediately after the taking-up, centrifugal again (centrifugal rotational speed is 10000rpm, time is 1.5min) after get supernatant liquor and transfer in the fresh centrifuge tube, namely obtain the fungal DNA extracting solution.
(3) utilize the fungal DNA extracting solution of fungi universal primer ITS1 and ITS4 to carry out pcr amplification, the system of pcr amplification is: the ITS1 primer of 10 * Taq buffer2 μ l, 10pmol and ITS4 primer each 1 μ l, fungal DNA extracting solution 2 μ l, 1U Taq DNA polymerase1 μ l, 2.5mM MgCl
21 μ l, 2mmol/LdNTPs1 μ l, aqua sterilisa complement to 20 μ l; Amplification condition is: 94 ℃ of 3min sex change, and 94 ℃ of 30s, 51 ℃ of 30s, 72 ℃ of 1min, 35 circulations, 72 ℃ of 7min extend.
(4) PCR product direct sequence is measured: amplified production is carried out agarose gel electrophoresis, and recycling sky root PCR reclaims test kit and reclaims the purpose fragment, delivers Shanghai Invitrogen company and carries out sequential analysis.
(5) NCBI blast sequence, judge which kind of fungi unknown fungi is: the nucleotides sequence that obtains is listed in the fungi sequence that NCBI website use blast search matches, matching length is the longest, and the fungi of sequence identity 100% is judged to be the fungi under the unknown fungi.
Above-mentioned fungi to be identified is selected from: the apple sheath blight fungus, the leaf muld of tomato bacterium, gaeumannomyces graminis, lawn coin pinta bacterium, verticillium dahliae, melon didymella bryoniae, the rice green smut bacterium, dosporium cucumerinumand its, corn is bent the spore germ, the sweet potato black rot bacterium, rice blast fungus, cucumber fusarium axysporum, peanut focal spot germ, the hami melon wilt, the Chinese cabbage alternaria, the Chinese sorghum rhizoctonia solani, the citrus scab bacterium, Chinese ephedra beading reaping hook fungi, the pepper anthracnose bacterium, cucumber anthracnose, or cereal reaping hook fungi;
The incubation time of above-mentioned 21 kinds of fungies is not wait in 1～5 day.
2, the Fig. 2 that the results are shown in Figure of pcr amplification has illustrated that present method is applicable to the Rapid identification of above-mentioned Different Crop pathogenic fungi.
The sequence alignment of above-mentioned 21 kinds of fungies the results are shown in following table 2, and the result shows that the reliability of the method for the Rapid identification pathogenic fungi of present embodiment reaches 100%.
The result of table 2 Different Crop pathogenic bacteria Rapid identification
| Crop pest | Pathogenic bacteria | PCR | Sequence blast |
| The apple sheath blight fungus | Physalospora?piricola | + | + |
| The leaf muld of tomato bacterium | Fulria?fulva | + | + |
| Gaeumannomyces graminis | Gaeumannomyces?graminis | + | + |
| Lawn coin pinta bacterium | Moellerodiscus?spp | + | + |
| Verticillium dahliae | Verticillium?dahliae | + | + |
| Melon didymella bryoniae | Didymella?Bryoniae | + | + |
| The rice green smut bacterium | Ustilaginoidea?virens | + | + |
| Dosporium cucumerinumand its | Cladosporium?cucumerinum | + | + |
| Corn is bent the spore germ | Curvularia?lunata | + | + |
| The sweet potato black rot bacterium | Ceratocystis?fimbriata | + | + |
| Rice blast fungus | Magnaporthe?oryzae | + | + |
| Cucumber fusarium axysporum | Fusarium?oxysporum.sp.cucumebrium | + | + |
| Peanut focal spot germ | Leptosphaerulina?crassiasca | + | + |
| The hami melon wilt | Fusarium?oxysporum?f.sp.Melonis | + | + |
| The Chinese cabbage alternaria | Alternaria?brassicae | + | + |
| The Chinese sorghum rhizoctonia solani | Rhizoctonia?solani?Kühn | + | + |
| The citrus scab bacterium | Sphaceloma?fawcetti?Jenk | + | + |
| Chinese ephedra beading reaping hook fungi | Fusarium?moniliforme | + | + |
| The pepper anthracnose bacterium | Colletotrichum?capsici | + | + |
| Cucumber anthracnose | Colletotrichum?lagenarium | + | + |
| Cereal reaping hook fungi | Fusarium?graminearum?Schw | + | + |
| The clear water contrast | ? | - | ? |
Annotate: "+" expression obtains purpose PCR fragment; "-" expression does not amplify the purpose fragment
The evaluation object of present embodiment comprises hypha,hyphae (radicula byssoidea), sclerotium and conidium, comprises the extraction of fungal DNA, the amplification of PCR specific fragment and three key steps such as sequencing of amplified fragments.
A kind of method of Rapid identification pathogenic fungi, concrete operations step and embodiment 1 are roughly the same, difference is: (1) cultivates fungi to be identified: the Aspergillus flavus, focal spot bacterium, alternaric bacteria, sickle-like bacteria and the white thin,tough silk bacterium that separate on the peanut are inoculated into respectively on the PDA substratum, 28 ℃ of dark cultivations were not waited in 1～4 day, grow to 2-3cm to bacterium colony, obtain the mycelia (being radicula byssoidea) of Aspergillus flavus, focal spot bacterium, alternaric bacteria, sickle-like bacteria and white thin,tough silk bacterium; Simultaneously the white thin,tough silk bacterium of peanut is inoculated on the PDA substratum 28 ℃ and secretly is cultured to the sclerotium that 10d obtains the white thin,tough silk bacterium of peanut; The aspergillus flavus bacterium is inoculated on czapek's solution (CZA) substratum conidium that 28 ℃ of dark culturing 5d obtain aspergillus flavus.
(2) microwave method is extracted fungal DNA: get fresh mycelia, sclerotium or conidium 1mg in the centrifuge tube of the 1.5ml of sterilization, 1 * TE damping fluid, the 50 μ l that add, shake after several seconds, be positioned in the microwave oven, 750W microwave 2min places 10min on ice fast after the taking-up, centrifugal again (centrifugal rotational speed is 10000rpm, time is 2min) after get supernatant liquor and transfer in the fresh centrifuge tube, namely obtain the fungal DNA extracting solution.
The form of above-mentioned fungi sees the following form 3, and 3, the Fig. 3 that the results are shown in Figure of pcr amplification has illustrated that this method is applicable to the DNA rapid extraction of mycelia, conidium or the sclerotium of peanut various pathogenic bacteria.
Sequence alignment to above-mentioned 5 kinds of fungies the results are shown in following table 3, the result shows, the fresh mycelia of fungi, sclerotium or the conidium of trace, at lower concentration 1 * TE damping fluid, after the short period of time microwave treatment, the DNA that all can obtain capacity carries out pcr amplification, and the pcr amplification band is clear, sequence blast compares demonstration, and the reliability of the method for the Rapid identification pathogenic fungi of present embodiment reaches 100%.
The qualification result of table 3 Different Kinds of Pathogens hypha,hyphae, conidium and sclerotium
| Pathogenic bacteria | Form | PCR | Sequencing |
| The aspergillus flavus bacterium | Conidium | + | + |
| Peanut focal spot bacterium | Mycelia | + | + |
| The peanut alternaric bacteria | Mycelia | + | + |
| The peanut sickle-like bacteria | Mycelia | + | + |
| The peanut sclerotium rolfsii bacterium | Mycelia | + | + |
| The peanut sclerotium rolfsii bacterium | Sclerotium | + | + |
Annotate :+purpose PCR fragment obtained;-expression does not amplify the purpose fragment
Obviously, above-described embodiment only is to be the example done of explanation clearly, and is not the restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here need not also can't give all embodiments exhaustive.And the apparent variation of therefore amplifying or change still are within the protection domain of the invention.
Claims (10)
1. the method for a Rapid identification pathogenic fungi is characterized in that comprising the steps:
(1) cultivates fungi to be identified, obtain radicula byssoidea, sclerotium or conidium;
(2) get above-mentioned radicula byssoidea, sclerotium or conidium and place damping fluid, carry out microwave thermal and shake after the broken wall treatment, place on ice, get supernatant liquor after centrifugal, be the fungal DNA extracting solution;
(3) utilize fungi universal primer ITS1 and ITS4 that above-mentioned fungal DNA extracting solution is carried out pcr amplification, amplified production carries out agarose gel electrophoresis, and recycling PCR reclaims test kit recovery purifying and obtains the purpose fragment;
(4) the above-mentioned purpose fragment is directly carried out sequential analysis, the nucleotides sequence that obtains is listed in the fungi sequence that NCBI website use blast search is complementary with it, and matching length is the longest, and sequence identity is that 100% fungi is judged to be the fungi of being identified under the fungi.
2. according to the described method of claim 1, it is characterized in that the described fungi to be identified of step (1) is selected from: the aspergillus flavus bacterium, the peanut sclerotium rolfsii bacterium, peanut focal spot germ, peanut reaping hook germ, the peanut alternaric bacteria, the apple sheath blight fungus, the leaf muld of tomato bacterium, gaeumannomyces graminis, lawn coin pinta bacterium, verticillium dahliae, melon didymella bryoniae, the rice green smut bacterium, dosporium cucumerinumand its, corn is bent the spore germ, the sweet potato black rot bacterium, rice blast fungus, cucumber fusarium axysporum, the hami melon wilt, the Chinese cabbage alternaria, the Chinese sorghum rhizoctonia solani, the citrus scab bacterium, Chinese ephedra beading reaping hook fungi, the pepper anthracnose bacterium, cucumber anthracnose, or cereal reaping hook fungi.
3. according to the described method of claim 1, it is characterized in that the described fungi of step (1) carries out flat board or slant culture at PDA substratum or czapek's solution.
4. according to the described method of claim 1, it is characterized in that the described fungi of step (1) 28 ℃ of dark cultivations on the PDA substratum grew to 2～3cm to bacterium colony in 1～5 day, obtain described radicula byssoidea; Or described fungi 28 ℃ of dark culturing 7～15 days on the PDA substratum, obtain described sclerotium; Or described fungi 28 ℃ of dark culturing 5～7 days on czapek's solution, obtain described conidium.
5. according to the described method of claim 1, it is characterized in that the described radicula byssoidea of step (2), sclerotium or conidial quality are 0.2～2mg.
6. according to the described method of claim 1, it is characterized in that the described damping fluid of step (2) is the TE damping fluid, its moiety is: 10mM Tris-HCl, 1mM EDTA, pH=8.0.
7. according to the described method of claim 1, it is characterized in that the shake microwave power of broken wall treatment of the described microwave thermal of step (2) is 750W, the time of microwave treatment is 30s～5min.
8. according to the described method of claim 1, it is characterized in that the described microwave thermal of step (2) shakes after the broken wall treatment, place 5～10min on ice immediately, carry out centrifugal then.
9. according to the described method of claim 1, it is characterized in that described centrifugally at room temperature carry out, centrifugal rotation speed is 10000rpm, the centrifugal time is 1～2min.
10. according to the described method of claim 1, it is characterized in that the system of described pcr amplification is: the ITS1 primer of 10 * Taq damping fluid, 2 μ l, 10pmol and ITS4 primer each 1 μ l, fungal DNA extracting solution 2 μ l, 1UTaq archaeal dna polymerase 1 μ l, 2.5mM MgCl
21 μ l, 2mmol/L dNTPs1 μ l, and aqua sterilisa complement to 20 μ l; Amplification condition is: 94 ℃ of 3min sex change, and 94 ℃ of 30s, 51 ℃ of 30s, 72 ℃ of 1min, 35 circulations, 72 ℃ of 7min extend.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310320242.4A CN103343168B (en) | 2013-07-26 | 2013-07-26 | Rapid identification method for pathogenic fungi |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310320242.4A CN103343168B (en) | 2013-07-26 | 2013-07-26 | Rapid identification method for pathogenic fungi |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103343168A true CN103343168A (en) | 2013-10-09 |
| CN103343168B CN103343168B (en) | 2015-03-11 |
Family
ID=49277993
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310320242.4A Active CN103343168B (en) | 2013-07-26 | 2013-07-26 | Rapid identification method for pathogenic fungi |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103343168B (en) |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103918518A (en) * | 2014-05-04 | 2014-07-16 | 湖南省水稻研究所 | Rice variety resource false smut resistance disease nursery identification method |
| CN104498474A (en) * | 2014-11-23 | 2015-04-08 | 云南省农业科学院农业环境资源研究所 | Method for direct extraction of pathogen DNA from pathogenic tissue of Graminaceous crop |
| CN104498473A (en) * | 2014-11-23 | 2015-04-08 | 云南省农业科学院农业环境资源研究所 | Method for rapid separation of pathogen DNA from rice blast single scab |
| CN105543392A (en) * | 2016-02-19 | 2016-05-04 | 河南农业大学 | Duplex-PCR detection method for maize kernel rot pathogen |
| CN105803051A (en) * | 2014-12-30 | 2016-07-27 | 徐立江 | Identification method of muskmelon black spot pathogen |
| CN106702014A (en) * | 2017-03-07 | 2017-05-24 | 福建省农业科学院植物保护研究所 | Peanut focal spot bacterial molecular detection primer and rapid detection method thereof |
| CN107796681A (en) * | 2017-09-28 | 2018-03-13 | 安徽信灵检验医学科技有限公司 | A kind of fungi fluorescent staining liquid and preparation method thereof |
| CN108048591A (en) * | 2017-12-08 | 2018-05-18 | 江汉大学 | A kind of Pyricularia grisea Race isolation and identification method |
| CN108148923A (en) * | 2018-02-22 | 2018-06-12 | 黑龙江省农业科学院植物脱毒苗木研究所 | The molecular detection primer and its detection method of potato plant tikka class disease alternaria solani sorauer pathogen |
| CN108660241A (en) * | 2018-04-27 | 2018-10-16 | 中国热带农业科学院环境与植物保护研究所 | A kind of specific primer and nested PCR detection method for detecting mango Bacterial Spot Pathogen |
| CN109355417A (en) * | 2018-11-14 | 2019-02-19 | 中国水稻研究所 | A kind of identification method of ustilaginoidea virens that is while producing five kinds of ustilaginoidea virens toxin |
| CN111893204A (en) * | 2020-08-14 | 2020-11-06 | 岭南师范学院 | Primer, kit and method for detecting high-resistance benzimidazole bactericide genotype E198A lawn scab |
| CN118109316A (en) * | 2024-04-28 | 2024-05-31 | 云南省林业和草原科学院 | Isolation and application of rhizosphere fungus muelleri YAFEF-2016 of pinus massoniana |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974640A (en) * | 2010-11-16 | 2011-02-16 | 杭州迪安医学检验中心有限公司 | Rapid Fungus detection kit and detection method using same |
| CN102586428A (en) * | 2012-01-13 | 2012-07-18 | 河南科技大学 | Detection method for pathogenicity of maize bacterial wilt source fusarium in maize seedling stage |
| CN102640671A (en) * | 2012-05-02 | 2012-08-22 | 湖北省农业科学院果树茶叶研究所 | Method for identifying Alternaria altemata (Fr.) Keissler resistance of pear germplasm |
| CN102732427A (en) * | 2010-09-29 | 2012-10-17 | 鄂尔多斯市普众生物技术有限公司 | Separation method of swainsonine-producing endophytic fungi in glabrous crazyweed |
-
2013
- 2013-07-26 CN CN201310320242.4A patent/CN103343168B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732427A (en) * | 2010-09-29 | 2012-10-17 | 鄂尔多斯市普众生物技术有限公司 | Separation method of swainsonine-producing endophytic fungi in glabrous crazyweed |
| CN101974640A (en) * | 2010-11-16 | 2011-02-16 | 杭州迪安医学检验中心有限公司 | Rapid Fungus detection kit and detection method using same |
| CN102586428A (en) * | 2012-01-13 | 2012-07-18 | 河南科技大学 | Detection method for pathogenicity of maize bacterial wilt source fusarium in maize seedling stage |
| CN102640671A (en) * | 2012-05-02 | 2012-08-22 | 湖北省农业科学院果树茶叶研究所 | Method for identifying Alternaria altemata (Fr.) Keissler resistance of pear germplasm |
Non-Patent Citations (5)
| Title |
|---|
| 冯岩等: "中国番木瓜枯萎病病原菌的首次鉴定", 《植物检疫》 * |
| 张晓利等: "4种黑曲霉基因组DNA提取方法的比较", 《中国真菌学杂志》 * |
| 张萍等: "苍耳柄锈菌三裂叶豚草专化型冬孢子基因组DNA快速提取方法的比较研究", 《菌物学报》 * |
| 晏立英等: "宜城市花生枯萎病病原的鉴定", 《湖北农业科学》 * |
| 曹永军等: "利用ITS1和ITS4通用引物扩增香蕉枯萎病菌核酸片段鉴定其生理小种", 《热带作物学报》 * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103918518A (en) * | 2014-05-04 | 2014-07-16 | 湖南省水稻研究所 | Rice variety resource false smut resistance disease nursery identification method |
| CN104498474A (en) * | 2014-11-23 | 2015-04-08 | 云南省农业科学院农业环境资源研究所 | Method for direct extraction of pathogen DNA from pathogenic tissue of Graminaceous crop |
| CN104498473A (en) * | 2014-11-23 | 2015-04-08 | 云南省农业科学院农业环境资源研究所 | Method for rapid separation of pathogen DNA from rice blast single scab |
| CN105803051A (en) * | 2014-12-30 | 2016-07-27 | 徐立江 | Identification method of muskmelon black spot pathogen |
| CN105543392A (en) * | 2016-02-19 | 2016-05-04 | 河南农业大学 | Duplex-PCR detection method for maize kernel rot pathogen |
| CN106702014A (en) * | 2017-03-07 | 2017-05-24 | 福建省农业科学院植物保护研究所 | Peanut focal spot bacterial molecular detection primer and rapid detection method thereof |
| CN107796681A (en) * | 2017-09-28 | 2018-03-13 | 安徽信灵检验医学科技有限公司 | A kind of fungi fluorescent staining liquid and preparation method thereof |
| CN108048591A (en) * | 2017-12-08 | 2018-05-18 | 江汉大学 | A kind of Pyricularia grisea Race isolation and identification method |
| CN108048591B (en) * | 2017-12-08 | 2021-07-02 | 江汉大学 | Separating and identifying method for small seeds of rice blast bacteria |
| CN108148923A (en) * | 2018-02-22 | 2018-06-12 | 黑龙江省农业科学院植物脱毒苗木研究所 | The molecular detection primer and its detection method of potato plant tikka class disease alternaria solani sorauer pathogen |
| CN108660241A (en) * | 2018-04-27 | 2018-10-16 | 中国热带农业科学院环境与植物保护研究所 | A kind of specific primer and nested PCR detection method for detecting mango Bacterial Spot Pathogen |
| CN109355417A (en) * | 2018-11-14 | 2019-02-19 | 中国水稻研究所 | A kind of identification method of ustilaginoidea virens that is while producing five kinds of ustilaginoidea virens toxin |
| CN111893204A (en) * | 2020-08-14 | 2020-11-06 | 岭南师范学院 | Primer, kit and method for detecting high-resistance benzimidazole bactericide genotype E198A lawn scab |
| CN118109316A (en) * | 2024-04-28 | 2024-05-31 | 云南省林业和草原科学院 | Isolation and application of rhizosphere fungus muelleri YAFEF-2016 of pinus massoniana |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103343168B (en) | 2015-03-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103343168B (en) | Rapid identification method for pathogenic fungi | |
| Shah et al. | Mycovirus-induced hypervirulence of Leptosphaeria biglobosa enhances systemic acquired resistance to Leptosphaeria maculans in Brassica napus | |
| CN108179115B (en) | Chrysanthemum endophytic spore shell bacteria and application thereof | |
| CN103740815B (en) | Primer for multi-PCR (Polymerase Chain Reaction) detection aiming at fusaria and application of primer | |
| CN105018351A (en) | Method for rapidly separating and identifying fungal endophytes of tobacco | |
| Sáenz-Mata et al. | Transcriptional regulation of Arabidopsis thaliana WRKY genes under interaction with beneficial fungus Trichoderma atroviride | |
| CN102719541A (en) | Method for analyzing Bacillus community structure in white spirit fermentation system | |
| CN101712984B (en) | Molecular specificity mark primer for authenticating mushroom strains 241 and 241-4 and detecting method | |
| CN102220320B (en) | Specific molecular marker of volvariella volvacea V23 strain as well as obtaining method and application thereof | |
| CN101805796B (en) | Primer for detecting the soybean phytophthora and kit and method thereof | |
| CN102399888A (en) | Molecular identification method for cochliobolus miyabeanus pathogens | |
| CN101086015A (en) | Mushroom 45 bacteria molecular specific mark and its obtaining method and uses | |
| CN109355222B (en) | Bacillus having antagonistic action on rice bacterial blight and separation and application thereof | |
| CN110982715A (en) | Purple spore bacterium gene engineering bacterium delta PlflbD with high spore yield as well as construction method and application thereof | |
| CN103882109B (en) | The molecular detecting method of sugarcane toppers maize ear rot pathogenic bacteria microspecies gx1 | |
| CN114276938B (en) | Paecilomyces EJKS strain, Fusarium E-9 strain and application thereof | |
| CN101086017B (en) | Mushroom 507 bacteria molecular specific mark and its obtaining method and uses | |
| CN110923153B (en) | Straw saprophytic bacteria and application thereof | |
| CN106434879A (en) | Method for quickly and efficiently detecting different strain mating types of cordyceps militaris | |
| CN106929572A (en) | A kind of specific primer for identifying diseases of mulberry fruits and the method that diseases of mulberry fruits identification is carried out using the primer | |
| CN109055620B (en) | Primer for simultaneously detecting CMV, SMV and BCMV in soybean and detection method thereof | |
| CN102732599A (en) | Method for detecting Vibrio vulnificus and Vibrio parahaemolyticus in seawater sample by duplex polymerase chain reaction | |
| Shafique et al. | Isolation and Identification of a novel leaf necrotic pathogen of Vigna radiata in Pakistan | |
| CN103898209A (en) | Method for detecting avirulence gene AVR-pik-B of magnaporthe grisea | |
| Kannahi et al. | Isolation, characterization and antagonistic effect of phosphate solubilizing microorganisms from Vigna radiata L. rhizospheric soil |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |